Nitric oxide (NO) and peroxynitrite, formed from NO and superoxide anion, have been implicated as mediators of neuronal damage following focal ischemia, but their molecular targets have not been defined. One candidate pathway is DNA damage leading to activation of the nuclear enzyme, poly(ADP-ribose) polymerase (PARP), which catalyzes attachment of ADP ribose units from NAD to nuclear proteins following DNA damage. Excessive activation of PARP can deplete NAD and ATP, which is consumed in regeneration of NAD, leading to cell death by energy depletion. We show that genetic disruption of PARP provides profound protection against glutamate-NO-mediated ischemic insults in vitro and major decreases in infarct volume after reversible middle cerebral artery occlusion. These results provide compelling evidence for a primary involvement of PARP activation in neuronal damage following focal ischemia and suggest that therapies designed towards inhibiting PARP may provide benefit in the treatment of cerebrovascular disease.

Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is important for N-methyl-D-aspartate (NMDA) receptor-dependent neurotransmitter release, neurotoxicity, and cyclic GMP elevations. The coupling of NMDA receptor-mediated calcium influx and nNOS activation is postulated to be due to a physical coupling of the receptor and the enzyme by an intermediary adaptor protein, PSD95, through a unique PDZ-PDZ domain interaction between PSD95 and nNOS. Here, we report the identification of a novel nNOS-associated protein, CAPON, which is highly enriched in brain and has numerous colocalizations with nNOS. CAPON interacts with the nNOS PDZ domain through its C terminus. CAPON competes with PSD95 for interaction with nNOS, and overexpression of CAPON results in a loss of PSD95/nNOS complexes in transfected cells. CAPON may influence nNOS by regulating its ability to associate with PSD95/NMDA receptor complexes.

Neuronal nitric-oxide synthase (nNOS) is subject to alternative splicing. In mice with targeted deletions of exon 2 (nNOS ⌬͞⌬ ), two alternatively spliced forms, nNOS␤ and ␥, which lack exon 2, have been described. We have compared localizations of native nNOS␣ and nNOS␤ and ␥ by in situ hybridization and immunohistochemistry in wild-type and nNOS ⌬͞⌬ mice. To assess nNOS catalytic activity in intact animals we localized citrulline, which is formed stoichiometrically with NO, by immunohistochemistry. nNOS␤ is prominent in several brain regions of wild-type animals and shows 2-to 3-fold up-regulation in the cortex and striatum of nNOS ⌬͞⌬ animals. The persistence of much nNOS mRNA and protein, and distinct citrulline immunoreactivity (cit-IR) in the ventral cochlear nuclei and some cit-IR in the striatum and lateral tegmental nuclei, indicate that nNOS␤ is a major functional form of the enzyme in these regions. Thus, nNOS␤, and possibly other uncharacterized splice forms, appear to be important physiological sources of NO in discrete brain regions and may account for the relatively modest level of impairment in nNOS ⌬͞⌬ animals.Nitric oxide (NO), formed by neuronal nitric-oxide synthase (nNOS) (1), has major signaling functions in the central and peripheral nervous system (2, 3). Alternative splicing of the nNOS gene is prominent with more than 10 differently spliced nNOS transcripts reported (4-10). The principal, exon 2-containing, form of nNOS (nNOS␣) accounts for the great majority of catalytic activity in the brain, as mice with targeted deletions of exon 2 (nNOS ⌬͞⌬ ) display an Ϸ95% reduction in NOS catalytic activity (11). Though NO has been implicated in development (12) and synaptic plasticity (13), these mice appear grossly normal, lack obvious histopathological abnormalities in the central nervous system, and reproduce effectively (11), suggesting that the residual NOS activity might protect the nNOS ⌬͞⌬ mutant mice from serious pathology. Two alternatively spliced forms of nNOS, ␤ and ␥, that lack exon 2 and thus persist in the nNOS ⌬͞⌬ mice, have a regional distribution that parallels the pattern of the residual activity (4).The human nNOS gene contains 29 exons of which all but exon 1 are translated to generate nNOS␣, a 150-kDa protein (1, 8). nNOS␤ and ␥, as described in mice, are generated by alternative splicing that skips exon 2 and employs different first exons, 1a and 1b, respectively (4) (Fig. 1). Translation of nNOS␤ is initiated at a CTG-initiation codon within exon 1a generating a 136-kDa NOS protein with six unique N-terminal amino acids. Translation of nNOS␥ is initiated at an ATGinitiation codon within exon 5 generating a truncated, 125-kDa form of nNOS␣. Both nNOS␤ and nNOS␥ lack the PSD-95͞ discs large͞ZO-1 homology domain (PDZ) domain that is localized within exon 2. This domain, also referred to as DHR or GLGF, mediates association of nNOS␣ with postsynaptic density protein 95 ref. 4). PSD-95 appears to anchor nNOS␣ to neuronal membranes in the vicinity of the Nmethyl-D-as...